General methods
The reaction progress was monitored by GC and TLC analyses. TLC was performed on Merck silica gel
plates 60 F254 and visualized using anisaldehyde/sulfuric acid reagent or UV absorption. GC-MS analysis was carried on Agilent Intuvo 9000 instrument using DB-5 column. The chiral GC analysis was obtained using chiral βeta-dex 120 column (30 m x 0.25 mm). IR spectra (in CHCl3 or neat) were recorded on FT-IR spectrometer. The fungusRhizopus oryzae, NCIM 1009, (ATCC 9363) culture was obtained from National Chemical Laboratory, Pune, India and culture media were obtained from Himedia, Mumbai, India. The cultures were grown aerobically at 27 °C and 150 rpm on orbital shaker (Scigenics Biotech, Orbitek). Citronellene and (S)-citronellol were obtained from S. H. Kelkar Company, Mumbai, India.Solvents were distilled and terpenes were vacuum distilled before use.
Culture media
The potato dextrose broth (PDB) containing potatoes infusion of 200 g /L, dextrose 20 g /L, in distilled water (pH 5.1± 0.2) was used. The cultures were maintained on Potato Dextrose Agar (PDA) containing potatoes infusions 200 g /L, dextrose 20 g /L, agar 20 g/L in distilled water (pH 5.1± 0.2).
Biotransformation
Rhizopus oryzae was grown on PDA slants. Inoculation was done by transferring a loopful of mycelium into the flask containing PDB (100 mL) in each flask. Cultures were allowed to grow in 10 flasks aerobically in the medium on an orbital shaker set at 100- 150 rpm and placed in a room maintained 27 ℃ for 72 h. Subsequently the substrate citronellol and citronellene (100 mg) dissolved in ethanol (1 mL) was added to each flask aseptically and the fermentation was continued for further 120 h and 48 h respectively with the agitation speed of 130 rpm.
After incubating the substrates withR.oryzae for 24 hrs,200ml of culture broth was removed eachafter every 24 hrs till day 5. The culture broth was filtered, extracted and then crude product was subjected to GC to monitor the rate of conversion. The optimum values of pH and temperature for biotransformation were found to be ~pH 5.5 at 27 ℃ for the mentioned incubation period.
Extraction of products
Extraction of bioconversion product was carried out after 8th day of fermentation period. The medium was filtered under vacuum and cells were separated. The broth containing the products was extracted with ethyl acetate (3×25mL). The organic layer containing the product was separated from aqueous layer. The organic layer was dried over anhydrous Na2SO4 for 2h. The solvent was removed under reduced pressure to give reaction products.
Purification of products
The reaction products were purified by silica gel column chromatography using a mixture of ethyl acetate: petroleum ether (60-80 °C) with increasing polarity. After elution the solvent was removed under reduced pressure to give pure molecules, which were identified with spectral analyses including FTIR, 1H &13C NMR and GC/EIMS analysis. The minor products were identified by GC/EIMS fragmentation matching the spectra from NIST library.The structures of products and yields obtained are given in Figure 2 and Table 1 respectively.
Products obtained from (S)-citronellol (1) included 7-hydroxycitronellol (3), rose oxide (5),Rose glycol (6), isopulegol (7),1,8- nonan-diol-8-methyl (8). artemisia triene (14) citronellyl- phenyl acetate (15) (±)-Citronellene (2) on similar treatment with R. oryzae was converted to 6,7-Dihydroxy-citronellene(4),Isopulegone (9), terpinene-4-ol (10),camphene (11),camphor (12) and α-pinene(13)
(Figure 2)
Spectral data of major products
7-Hydroxycitronellol(3)
Colourless oil; Yield 22.5%, [α]D -4.3 (c 1.5 MeOH), -4.19 (c 0.37 CHCl3), Chiral GC (rt) 28.05 min; GC/MS m/z 159 [M-Me]+ , 141 [159–H2O]+ , 123, 98, 83, 71, 59 (100%), 55, 53, 43. 159; IR νmax: 3360, 1470, 1381 Cm-1; 1H- NMR: δ 0.87 (d, J= 6 Hz, 3H); 1.17 (s, 6H), 1.27-1.41 (m, 3H), 1.5-1.62 (m, 1H), 3.60- 3.68 (m, 2H) ; 13C- NMR: δ 19.7; 21.7, 29.3, 29.4, 29.5, 37.6, 39.8, 44.1, 61.0, 71.3. Spectral data of compound 3fully agreed with earlier report [Nankai et al. 1997, Kutner et al. 1995]. (Fig. 5, Fig 6)
6,7-Dihydroxy-citronellene-diol 4
Colourless oil; Yield 23 %, [α]D -12.7 (c 0.384 MeOH); Chiral GC (rt) 24.09 & 24.32 min; GC/MS m/z 154 [M-H2O]+ , 139 [154–Me]+ , 121, 114, 96, 95, 81, 71, 59 (100%), 55, 53, 43; IR νmax: 3420, 1638, 1458, 1383, 1363 cm-1; 1H-NMR: δ 1.02 (3H, d, J= 6.9 Hz) [1.01 (3H, d, J= 6.9 Hz)]; 1.14 (3H, s), 1.20 (3H, s), 1.27-1.41 (3H, m), 1.5-1.72 (1H, m), 3.35 (1H, dd, J= 10, 2 Hz); [3.32 (1H, dd, J= 10, 1.5 Hz)]; 4.92 (1H, dd, 10.3, 1.4), 4.98 (1H, dd, 17, 1.2), 5.70 (1H, ddd, 17, 10, 7.5) ; 13C-NMR: δ 20.7 (20.3); 23.3, 26.7, 29.4 (29.6), 33.6 (33.9), 37.9 (38.1), 73.4, 78.6 (79.1), 112.9 (113.2), 144.5 (144.7). Spectral data fully agreed with earlier report [Miyazawa et al. 2009] where (4) was metabolite produced by a Larvae obtained in 3.1% yield. (Fig.8, Fig. 9)
GC/MS data of minor products
Rose oxide (5)- m/z 154, 139 (100 %), 91, 83, 77, 69, 55, 41
Rose glycol (6)-m/z 121 (M+ - 79), 99, 86, 71(100 %), 55.41.
Isopulegol (7)- m/z 154, 136, 121, 109, 95, 81, 69, 55, 41(100 %).
1,8- Nonan-diol,8-methyl (8)-m/z 159, 141, 123, 98.1, 83.1, 70.1, 59.1(100%), 53, 41.
Isopulegone (9)- m/z 152, 137, 109, 95, 81 (100 %), 67, 53, 41.
Terpinene-4-ol (10)- m/z 154, 111, 93, 83, 71, 67, 55(100 %), 43.
Camphene (11)- m/z 136, 121, 106, 93(100 %), 79, 67, 53, 43.
Camphor (12)- m/z 153 (M+ +1), 137, 121, 108, 95(100 %), 90, 81, 69, 55, 40
α-Pinene (13)-m/z 136, 121, 105, 93(100%), 77, 67, 58, 53, 41
Artemisia triene (14)- m/z 136, 121, 107, 93(100 %), 79, 67, 55, 43.
Citronellyl- phenyl acetate (15)- m/z 208 (M+ -66), 192, 138.1, 123, 109, 95, 81 (100 %), 69, 55, 41.
(Figure- 5,6,7,8,9,10)